PURPOSE: To establish polyhexamethylene biguanide (PHMB) as an effective treatment for Aspergillus keratitis in a novel murine model. To determine the ability of the calcineurin inhibitors tacrolimus (FK506) and cyclosporine A (CSA) to enhance the activity of PHMB, amphotericin B (AMB), and voriconazole (VCZ) against Aspergillus keratitis. IN VITRO STUDIES: Broth antifungal susceptibility tests were performed with PHMB, AMB, VCZ, and FK506, individually and in combination against Aspergillus fumigatus. Minimum inhibitory concentrations (MIC) and fractional inhibitory concentration index (FICI) values were used to analyze antifungal activity. In vivo studies: A novel murine model was created to establish Aspergillus keratitis. Infected mice were randomly assigned to treatment groups receiving saline, CSA, AMB, VCZ, PHMB, AMB+CSA, VCZ+CSA, or PHMB+CSA. An ophthalmologist blinded to the treatment groups assessed disease severity daily based on a grading scale. The mean end change in disease score was compared between groups. IN VITRO STUDIES: FK506 in combination with PHMB, VCZ, or AMB enhanced fungal growth inhibition. FICI values showed an additive effect between FK506 and PHMB, AMB, or VCZ. PHMB monotherapy eliminated Aspergillus growth starting at 4 μg/mL. In vivo studies: All treatment groups showed a significant improvement in disease score compared to the control group. CSA significantly worsened VCZ activity against Aspergillus keratitis. CONCLUSIONS: PHMB is an effective inhibitor of Aspergillus growth. Further investigation of the role of calcineurin inhibitors in the treatment for Aspergillus keratitis is warranted.